Patent application title: Creatine compositions for skin treatment

Abstract:

The present invention relates to the use of creatine compounds, such as,
for example, creatine monohydrate, creatine pyruvate and creatine
ascorbate, for the treatment of skin.

Claims:

1. A method for treating uneven pigmentation in a subject's skin,
comprising administering to said subject an amount of a creatine compound
effective to modulate tyrosinase, wherein said amount of the creatine
compound is at least between about 0.0001-10% by weight, such that said
subject's skin is treated for uneven pigmentation.

2. The method of claim 1, wherein said creatine compound is an inhibitor
of tyrosinase.

3. The method of claim 2, wherein said inhibitor inhibits tyrosinase by at
least about 25% at a concentration of about 5 μg/mL of said creatine
compound.

4. The method of claim 3, wherein said creatine compound is creatine
ascorbate.

5. The method of claim 2, wherein said inhibitor inhibits tyrosinase by at
least about 100% at a concentration of about 500 g/mL of said creatine
compound.

6. The method of claim 2, wherein said inhibitor inhibits tyrosinase by at
least about 10% at a concentration of about 50 g/mL of said creatine
compound.

7. The method of claims 5 or 6, wherein said creatine compound is creatine
pyruvate.

8. A method for quenching free radicals in a subject's skin, comprising
administering to said subject an amount of a creatine compound effective
to provide an antioxidant effect, wherein said amount of the creatine
compound is at least between about 0.0001 and 10% by weight, such that
free radicals are quenched.

11. The method of claims 9 or 10, wherein said creatine compound is
creatine ascorbate.

12. A method for preserving a subject's skin, comprising administering to
said subject an amount of a creatine compound effective to inhibit lipid
peroxidation, wherein said amount of the creatine compound is at least
about between about 0.0001 and 10% by weight, such that said subject's
skin is preserved.

13. The method of claim 11, wherein said creatine compound inhibits by at
least about 20% of lipid peroxidation at a concentration of about 500
g/mL of said creatine compound.

14. The method of claim 13, wherein said creatine compound is creatine
ascorbate.

15. A method for treating aging of a subject's skin, comprising
administering to said subject an amount of a creatine compound effective
to modulate mitochondrial metabolism, wherein said amount of the creatine
compound is at least between about 0.0001 and 10% by weight, such that
said aging of a subject's skin is treated.

17. The method of claim 16, wherein said creatine compound stimulates
mitochondrial metabolism by at least about 15% at a concentration of
about 100 g/mL of the creatine compound.

18. The method of claim 15, wherein said creatine compound stimulates
mitochondrial metabolism by at least about 25% at a concentration of
about 1000 g/mL of the creatine compound.

19. The method of claims 16 or 17, wherein said creatine compound is
creatine monohydrate.

20. A method of treating a subject's skin for UV irradiation stress,
comprising administering an effective amount of a creatine compound to
said subject, wherein said effective amount of a creatine compound is a
least between about 0.0001 and 10% by weight, such that the subject's
skin is treated.

21. A method of modulating carcinogenic stress in a subject's skin,
comprising administering an effective amount of a creatine compound to
said subject, such that said carcinogenic stress is modulated.

22. The method of claim 21, wherein said creatine compound protects
against or inhibits carcinogenic stress.

23. The method of claim 20 or 21, wherein said creatine compound is
creatine monohydrate, creatine pyruvate or creatine ascorbate.

24. A method for modulating collagen levels in a subject's skin,
comprising administering to said subject of a at least between about
0.0001 and 10% by weight of a creatine compound, such that collagen
levels in said subject's skin are modulated.

40. A cosmetic composition comprising an effective amount of a creatine
compound, a cosmetically acceptable carrier, and one or more cosmetic
adjuvants, wherein said effective amount of a creatine compound is at
least about between about 0.0001-10% by weight.

41. The composition of claim 40, wherein said creatine compound is
selected from the group consisting of creatine monohydrate, creatine
ascorbate and creatine pyruvate.

42. The composition of claim 41, wherein said cosmetic composition is
formulated as a foundation.

45. The composition of claim 41, wherein said cosmetic adjuvant is a dye,
opacifier, pigment, or a perfume.

46. The composition of claim 41, further comprising surfactants,
emulsifiers, gelling agents, penetration enhancers, solubilizers, skin
protectants, and sunscreen agents 47. A packaged composition comprising
an effective amount of a creatine compound, a cosmetically acceptable
carrier, and one or more cosmetic adjuvants, wherein said effective
amount of a creatine compound is at least between about 0.0001 and 10% by
weight.

48. The packaged composition of claim 47, further comprising instructions
for use.

49. The method of any one of claims 1, 8, 12, 15, 20, and 24 wherein said
effective amount is at least between about 0.001 and 1% by weight.

50. The method of any one of claims 1, 8, 12, 15, 20 and 24, wherein said
effective amount is at least between about 0.01 and 0.5% by weight.

51. The composition of claim 40, wherein said effective amount is at least
between about 0.001 and 1% by weight.

52. The composition of claim 40, wherein said effective amount is at least
between about 0.01 and 0.5% by weight.

53. The packaged composition of claim 47, wherein said effective amount is
at least between about 0.001 and 1% by weight.

54. The packaged composition of claim 47, wherein said effective amount is
at least between about 0.01 and 0.5% by weight.

[0002]The creatine kinase/creatine phosphate energy system is only one
component of an elaborate energy-generating system found in tissue with
high and fluctuating energy requirements. The components of the creatine
energy system include the enzyme creatine kinase, the substrates creatine
and creatine phosphate, and the transporter of creatine. The reaction
catalyzed by creatine kinase is: MgADP+PCr.sup.=+H.sup.+ MgATP.sup.=+Cr.
Some of the functions associated with this system include efficient
regeneration of energy in cells with fluctuating and high energy demands,
energy transport to different parts of the cell, phosphoryl transfer
activity, ion transport regulation, and involvement in signal
transduction pathways.

[0003]Creatine is a compound which is naturally occurring and is found in
mammalian brain and other excitable tissues, such as skeletal muscle,
retina and heart. Its phosphorylated form, creatine phosphate, also is
found in the same organs and is the product of the creatine kinase
reaction utilizing creatine as a substrate. Creatine phosphate is one of
the highest energy generating compounds in the cell and creatine is an
excellent stimulant of oxidative phosphorylation and high energy
production. Creatine has been extensively used by body builders as a
means of stimulating energy production in the skeletal muscle. Creatine
and creatine phosphate can be synthesized relatively easily and are
believed to be non-toxic to mammals. Creatine, creatine phosphate and the
enzymes that utilize them as substrates (i.e. the creatine kinases)
represent an efficient system for the rapid regeneration of energy.
Kaddurah-Daouk et al. (WO 92/08456; WO 90/09192; U.S. Pat. No. 5,321,030;
and U.S. Pat. No. 5,324,731) describe methods of inhibiting the growth,
transformation and/or metastasis of mammalian cells using related
compounds. Examples of compounds described by Kaddurah-Daouk et al.
include cyclocreatine, b-guandidino propionic acid, homocyclocreatine,
1-carboxymethyl-2-iminohexahydropyrimidine, guanidino acetate and
carbocreatine. These same inventors have also demonstrated the efficacy
of such compounds for combating viral infections (U.S. Pat. No.
5,321,030). Elebaly in U.S. Pat. No. 5,091,404 discloses the use of
cyclocreatine for restoring functionality in muscle tissue. Cohn in PCT
publication No. WO 94/16687 described a method for inhibiting the growth
of several tumors using creatine and related compounds. Kaddurah-Daouk et
al. (WO 96/14063) reported on the neuroprotective effect of creatine
compounds especially against neurodegenerative diseases such as
Huntington's, Parkinson's, ALS, Alzheimer's.

[0004]Aging involves death of cells or cell dysfunction due to production
of free radicals, oxidative damage and energy depletion due to
mitochondrial dysfunction. Harman (1988) linked senescence or death to
the injurious effects of free radicals arising from the one-electron
reduction of oxygen during metabolism. There has been an inverse
relationship between auto-oxidation rate in different animal species and
life expectancy in the same species (Cutler 1985; Sohal 1995).
Mitochondria are the major source of oxygen radicals through the
respiratory chain and are also deeply affected by reactive oxygen species
(ROS), resulting in serious risks to their function. Mitochondrial
dysfunction could result in defects in electron transport, oxidative
phosphorylation and energy production resulting in cell damage and
ultimately cell death.

SUMMARY OF THE INVENTION

[0005]In one embodiment, the invention pertains, at least in part, to a
method for treating uneven pigmentation in a subject's skin by
administering to a subject an amount of a creatine compound effective to
modulate tyrosinase, in which the amount of the creatine compound is at
least between about 0.0001 and 10% by weight.

[0006]In another embodiment, the invention pertains, at least in part, to
a method for quenching free radicals in a subject's skin, by
administering to a subject an amount of a creatine compound effective to
provide an antioxidant effect, in which the amount of the creatine
compound is at least between about 0.0001 and 10% by weight.

[0007]In yet another embodiment, the invention pertains, at least in part,
to a method for preserving a subject's skin, by administering to a
subject an amount of a creatine compound effective to inhibit lipid
peroxidation, in which the amount of the creatine compound is at least
between about 0.0001 and 10% by weight.

[0008]In a further embodiment, the invention pertains, at least in part,
to a method for treating aging of a subject's skin, by administering to a
subject an amount of a creatine compound effective to modulate
mitochondrial metabolism, in which the amount of the creatine compound is
at least between about 0.0001 and 10% by weight.

[0009]In one embodiment, the invention pertains, at least in part, to a
method of treating a subject's skin for UV irradiation stress by
administering an effective amount of a creatine compound to the subject,
wherein said effective amount of a creatine compound is a least between
about 0.0001 and 10% by weight.

[0010]In one embodiment, the invention pertains, at least in part, to a
method of modulating carcinogenic stress in a subject's skin, by
administering an effective amount of a creatine compound to a subject.

[0011]In yet another embodiment, the invention pertains, at least in part,
to a method for modulating collagen levels in a subject's skin,
comprising administering to said subject of a at least between about
0.0001 and 10% by weight of a creatine compound, such that collagen
levels in said subject's skin are modulated.

[0012]In a further embodiment, the invention pertains, at least in part,
to a method of treating inflammation in a subject, by administering an
amount of a creatine compound effective to modulate metalloproteinase.

[0013]In one embodiment, the invention pertains, at least in part, to a
cosmetic composition comprising an effective amount of a creatine
compound, a cosmetically acceptable carrier, and one or more cosmetic
adjuvants, in which the effective amount of the creatine compound is at
least between about 0.0001 and 10% by weight.

[0014]In yet another embodiment, the invention further pertains, at least
in part, to a packaged composition comprising an effective amount of a
creatine compound, a cosmetically acceptable carrier, and one or more
cosmetic adjuvants, in which the effective amount of the creatine
compound is at least between about 0.0001 and 10% by weight.

[0023]The methods of the present invention generally comprise
administering to a subject an effective amount of a creatine compound or
compounds.

[0024]The term "creatine compound" includes creatine, derivatives of
creatine, creatine-ligand compounds and pharmaceutically acceptable salts
of creatine. Examples of derivatives of creatine include cyclocreatine,
creatine phosphate, cyclocreatine phosphate, etc. Examples of creatine
derivatives are described in U.S. Pat. No. 6,242,491 B1, incorporated
herein by reference.

[0025]Creatine (also known as N-(aminoiminomethyl)-N-methylglycine;
methylglycosamine or N-methyl-guanido acetic acid) is a compound of
formula (I):

[0026]In a further embodiment, the creatine compound is creatine
ascorbate, creatine pyruvate, or creatine monohydrate.

[0027]In a further embodiment, the creatine compound is a creatine-ligand
compound, wherein the creatine-ligand compound has a ratio of between
about 1:1 creatine to ligand and about 10:1 creatine to ligand. The term
"ligand" includes a compound in which creatine is bound to another atom
or molecule through covalent or electrostatic interactions. The ratio of
creatine to the ligand can be, for example, about a 1:1 ratio, about a
2:1 ratio, about a 3:1 ratio, about a 4:1 ratio, about a 5:1 ratio, about
a 6:1 ratio, about a 7:1 ratio, about an 8:1 ratio, about a 9:1 ratio or
about a 10:1 ratio. The ratio of creatine to the ligand can also be any
ratio in which creatine is bound to the ligand through covalent or
electrostatic interactions. In one embodiment, the creatine-ligand
compound has a ratio of between about 3:1 creatine to ligand and about
6:1 creatine to ligand.

[0029]In one embodiment, the ligand is a water-soluble vitamin. The term
"water-soluble vitamin" includes those vitamins which dissolve easily in
water, such as vitamin C (ascorbic acid) and the B-complex vitamins. The
B-complex vitamins may include vitamin B1 (thiamine), B2
(riboflavin), B3 (niacin), B5 (pantothenic acid), B6
(pyridoxine), B7 (biotin), B9 (folic acid), and B12
(cyanocobalamin).

[0030]In another embodiment, the ligand is selected from the group
consisting of cinnamate, lactate, glycolate, malate, mandelate,
ascorbate, phytate, citrate, hydroxycitrate, aleurate, salicylate and
hyaluronate. In one particular embodiment, the ligand is ascorbate.

[0031]The term "subject" includes living organisms, such as humans, dogs,
cats, horses, goats, cows, pigs, rodents, monkeys, gorillas, bears,
chimpanzees and cattle. The term "subject" further is intended to include
transgenic species.

[0032]In one embodiment, the invention pertains to a method for modulating
the melanin synthesis pathway. In humans, melanin is found in skin, hair,
the pigmented epithelium underlying the retina, the medulla and zona
reticularis of the adrenal gland, the stria vascularis of the inner ear,
and in pigment bearing neurons of certain deep brain nuclei such as the
locus ceruleus and the substantia nigra. Melanin is believed to be the
primary determinant of human skin color. Dermal melanin is produced by
melanocytes, which are found in the stratum basale of the epidermis.
Although human beings generally possess a similar concentration of
melanocytes in their skin, the melanocytes in some individuals and ethnic
groups more frequently or less frequently express the melanin-producing
genes, thereby conferring a greater or lesser concentration of skin
melanin. Some individual animals and humans have no or very little
melanin in their bodies, which is a condition known as albinism.

[0033]Because melanin is an aggregate of smaller component molecules,
there are a number of different types of melanin with differing
proportions and bonding patterns of these component molecules. Both
pheomelanin and eumelanin are found in human skin and hair, but eumelanin
is the most abundant melanin in humans, as well as the form most likely
to be deficient in albinism. The precise nature of eumelanin's molecular
structure is the object of study. Eumelanin is found in hair and skin,
and colors hair grey, black, yellow, and brown. In humans, it is more
abundant in peoples with dark skin. There are two different types of
eumelanin, which are distinguished from each other by their pattern of
polymer bonds. The two types are black eumelanin and brown eumelanin. A
small amount of black eumelanin in the absence of other pigments causes
grey color. A small amount of brown eumelanin in the absence of other
pigments causes yellow (blond) color. Pheomelanin is also found in hair
and skin and is more abundant in fair-skinned humans. Pheomelanin imparts
a pink to red hue and, thus, is found in particularly large quantities in
red hair. Pheomelanin is particularly concentrated in the lips, nipples,
glans of the penis, and vagina. Pheomelanin also may become carcinogenic
when exposed to the ultraviolet rays of the sun. Chemically, pheomelanin
differs from eumelanin in that its oligomer-structure incorporates the
amino acid L-cysteine, as well as DHI and DHICA units.

[0034]The first step in the biochemical synthesis of the components of
melanin (both eumelanins and pheomelanis) in the body is the
tyrosinase-mediated transformation of tyrosine to DOPA
(3,4-dihydroxy-L-phenylalanine) and dopaquinone. Dopaquinone can then
combine with cysteine to form either 5-S-cysteinyldopa or
2-S-cysteinyldopa, which, via a benzothiazine intermediate, forms
pheomelanin. Alternatively, dopaquinone can be converted to
leucodopachrome and dopachrome, which can subsequently be converted to
either 5,6-dihydroxyindole-2-carboxylic acid or 5,6-dihydroxyindole.
These two compounds are then converted into quinine, followed by the
production of eumelanin.

[0035]The language "melanin synthesis pathway" includes any biochemical
pathway for the synthesis and/or upregulation of any of the components of
melanin in the body, as described above. The language "modulating the
melanin synthesis pathway" includes adjusting the melanin synthesis
pathway or keeping the melanin synthesis pathway in proper measure or
proportion. In one embodiment, the language "modulating the melanin
synthesis pathway" includes upregulating the melanin synthesis pathway or
any of the components of the melanin synthesis pathway. In another
embodiment, the language "modulating the melanin synthesis pathway"
includes inhibiting the melanin synthesis pathway or any of the
components of the melanin synthesis pathway.

[0036]Skin disorders that may be caused by a disturbance of the melanin
synthesis pathway include, for example, albinism and uneven pigmentation
(e.g., age spots, liver spots, freckles, moles, etc.) and skin cancer
(e.g., melanoma).

[0037]The language "treating uneven pigmentation" includes the prevention,
alleviation or amelioration of one or more symptoms of uneven
pigmentation. For example, treating uneven pigmentation may refer to the
fading or disappearance of the uneven pigmentation.

[0038]In one embodiment, the invention pertains to the treatment of uneven
pigmentation in the skin of a subject by administering to the subject an
amount of a creatine compound effective to modulate tyrosinase (e.g., the
conversion of tyrosine to DOPA and dopaquinone). The language "an amount
of a creatine compound effective to modulate tyrosinase" includes the
amount of a creatine compound necessary to upregulate or inhibit
tyrosinase activity.

[0039]In one embodiment, the amount of creatine compound effective to
modulate tyrosinase is at least between about 0.0001% to 10% by weight.
More particularly, the amount of the creatine compound necessary to
modulate tyrosinase may be about 0.0001%, 0.0002%, 0.0003%, 0.0004%,
0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%,
0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%, 0.009%, 0.01%, 0.02%,
0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and
10% by weight, or a range thereof. In a further embodiment, the amount of
creatine compound effective to modulate tyrosinase is at least between
about 0.001% and 1% by weight or between about 0.01% and 0.5% by weight.

[0041]In a one particular embodiment, the creatine compounds, such as
creatine ascorbate and creatine pyruvate, inhibit tyrosinase by at least
about 25% at a concentration of about 5 μg/mL, at least about 100% at
a concentration of about 500 μg/mL, or by at least about 10% at a
concentration of about 50 μg/mL. The inhibition of tyrosinase can be
determined using the methods described in Example 7.

[0042]In one embodiment, the invention pertains to a method of treating
the skin of a subject for oxidative stress. The language "oxidative
stress" includes the imbalance between free radicals and antioxidants,
which causes damage to the cells of a subject. Free radicals are atomic
or molecular species with unpaired electrons on an otherwise open shell
configuration. These unpaired electrons are highly reactive and the
amount of free radicals typically increases during environmental stress.
Typically, cells are able to defend themselves against free radical
damage through the use of enzymes such as superoxide dismutases and
catalases.

[0043]The effects of free radicals on cell metabolism have been well
documented in a variety of species, and include roles in programmed cell
death, apoptosis, cancer and inflammatory responses. In addition, free
radicals play an important role in the death and regeneration of skin
cells.

[0044]In one particular embodiment, the invention pertains to a method for
quenching free radicals in a subject's skin by administering to the
subject an amount of a creatine compound effective to provide an
antioxidant effect. The language "quenching free radicals," includes the
suppression of free radicals, inhibition of the generation of free
radicals or the treating the damage caused by free radicals. The language
"antioxidant effect" includes the effect of quenching free radicals
(e.g., the effect of suppressing free radicals, the effect of inhibiting
the generation of free radicals or the effect of treating the damage
caused by free radicals).

[0045]The language "an amount of a creatine compound effective provide an
antioxidant effect" includes the amount of a creatine compound necessary
to quench at least a portion of the free radicals (e.g., suppression of
free radicals, inhibition of the generation of free radicals or the
treating the damage caused by free radicals), preferably most, if not
all, of the free radicals in the subject's skin. The antioxidant effect
of the creatine compounds of the invention can be determined using the
method described in Example 4.

[0046]In one embodiment, the amount of creatine compound effective to
quench free radicals is at least between about 0.0001% to 10% by weight.
More particularly, the amount of the creatine compound necessary to
modulate tyrosinase may be about 0.0001%, 0.0002%, 0.0003%, 0.0004%,
0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%,
0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%, 0.009%, 0.01%, 0.02%,
0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and
10% by weight, or a range thereof. In a further embodiment, the amount of
creatine compound effective to quench free radicals is at least between
about 0.001% and 1% by weight or between about 0.01% and 0.5% by weight.

[0047]One method for determining the antioxidant effect of compound is
measuring the oxygen radical absorbance capacity (ORAC) of that compound
(see Example 4). The units of the ORAC are micromole Trolox equivalents
(TE) per gram of compound. The higher the Trolox equivalents, the greater
the capacity of the compound to absorb free radicals. In one embodiment,
the creatine compound exhibits at least about 5300 μM Trolox
equivalents/gram. In another embodiment, the creatine compound exhibits a
larger ORAC than magnesium ascorbyl phosphate. In yet another embodiment,
the creatine compound exhibits an antioxidant activity greater than
magnesium ascorbyl phosphate. In yet another embodiment, the creatine
compound is creatine ascorbate.

[0048]In one embodiment, the invention pertains to a method for preserving
the skin of a subject by administering to a subject an amount of a
creatine compound effective to inhibit oxidation of the skin. In another
embodiment, the invention pertains to a method for preserving the skin of
a subject by administering to a subject an amount of a creatine compound
effective to inhibit lipid peroxidation. The language "preserving the
skin" includes maintaining the skin or protecting the skin from harm.

[0049]The term "lipid peroxidation" includes the oxidative degradation of
lipids in cell membranes, resulting in cell damage.

[0050]The language "an amount of a creatine compound effective to inhibit
lipid peroxidation" includes the amount of a creatine compound necessary
to inhibit lipid peroxidation. The inhibition of lipid peroxidation can
be determined using the method described in Example 1.

[0051]In one embodiment, the amount of creatine compound effective to
inhibit lipid peroxidation is at least between about 0.0001% to 10% by
weight. More particularly, the amount of the creatine compound necessary
to inhibit lipid peroxidation may be about 0.0001%, 0.0002%, 0.0003%,
0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%,
0.003%, 0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%, 0.009%, 0.01%,
0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, and 10% by weight, or a range thereof. In a further embodiment, the
amount of creatine compound effective to inhibit lipid peroxidation is at
least between about 0.001% and 1% by weight or between about 0.01% and
0.5% by weight.

[0053]Mitochondria play an important role in many metabolic tasks, such
as, for example apoptosis-programmed cell death, energy conversion,
cellular proliferation and regulation of the cellular redox state, and
regulation of mitochondrial metabolism is an important method of treating
aging of the skin.

[0054]In one embodiment, the invention pertains to a method for treating
aging in the skin of a subject by administering to a subject an amount of
a creatine compound effective to modulate mitochondrial metabolism. The
language "modulate mitochondrial metabolism" includes adjusting the
mitochondrial metabolism or keeping the mitochondrial metabolism in
proper measure or proportion. In one embodiment, modulating mitochondrial
metabolism includes inhibiting mitochondrial metabolism. In another
embodiment, modulating mitochondrial metabolism includes stimulating or
increasing mitochondrial metabolism.

[0055]The language "an amount of a creatine compound effective to modulate
mitochondrial metabolism" includes the amount of a creatine compound
necessary to inhibit, increase or stimulate mitochondrial metabolism.

[0056]In one embodiment, the amount of creatine compound effective to
modulate mitochondrial metabolism is at least between about 0.0001% to
10% by weight. More particularly, the amount of the creatine compound
necessary to modulate mitochondrial metabolism may be about 0.0001%,
0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%,
0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%,
0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%,
0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, and 10% by weight, or a range thereof. In a further
embodiment, the amount of creatine compound effective to modulate
mitochondrial metabolism is at least between about 0.001% and 1% by
weight or between about 0.01% and 0.5% by weight.

[0058]In one particular embodiment, the creatine compound stimulates
mitochondrial metabolism. In another embodiment, the creatine compounds,
such as creatine monohydrate, stimulate mitochondrial metabolism by at
least about 15% at a concentration of about 100 μg/mL or by at least
about 25% at a concentration of about 1000 μg/mL of the creatine
compound. The stimulation of mitochondrial metabolism can be measured
using the method described in Example 3.

[0059]In one embodiment, the invention pertains to a method of treating UV
irradiation stress in a subject's skin by administering an effective
amount of a creatine compound to said subject. The term "treating UV
irradiation" includes preventing UV radiation or ameliorating the effects
of UV radiation. UV radiation can refer to both UVA radiation and UVB
radiation.

[0060]In another embodiment, the invention pertains to a method of
modulating carcinogenic stress in a subject's skin by administering an
effective amount of a creatine compound to the subject. The term
"modulating carcinogenic stress" includes the inhibition of carcinogenic
stress or protection against carcinogenic stress. The language
"carcinogen stress" includes any stimulus or circumstance that may induce
or cause cancer, such as skin cancer. Examples of stimuli include
carcinogenic chemicals, environmental factors and aging. In one
embodiment, the stimulus is not sun radiation, UVA, UVB, or UVC
radiation. In one embodiment, the cancer may be, for example, basal cell
carcinoma, squamous cell carcinoma, malignant melanoma,
dermatofibrosarcoma protuberans, Merkel cell carcinoma or Kaposi's
sarcoma.

[0062]The term "effective amount of a creatine compound" includes the
amount of a creatine compound necessary to alleviate, prevent or
ameliorate one or more symptoms that the administration of the creatine
compound is attempting to treat.

[0063]In one embodiment, an effective amount of creatine compound is at
least between about 0.0001% to 10% by weight. More particularly, an
effective amount of creatine compound may be about 0.0001%, 0.0002%,
0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%,
0.002%, 0.003%, 0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%, 0.009%,
0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%,
0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, and 10% by weight, or a range thereof. In a further
embodiment, the effective amount of creatine compound is at least between
about 0.001% and 1% by weight or between about 0.01% and 0.5% by weight.

[0064]In another embodiment, the invention pertains to a method for
treating photodamage and aging in a subject's skin by administering to a
subject an amount of a creatine compound effective to modulate collagen
levels. The term "photodamage" and "photoaging" may be used to describe
chronic changes in the appearance and function of the skin caused by
repeated sun exposure rather than by the passage of time (the latter
called intrinsic or chronologic aging). The term "acute photodamage"
includes sunburn. Photodamage may also be called dermatoheliosis.
Symptoms of photodamage include, for example, skin changes of fine and
coarse wrinkles, roughness, laxity, mottled pigmentation, actinic
lentigines, actinic keratoses, leathery texture/coarseness,
scaling/xerosis, sallowness, and telangiectasia, as well as cancer. In
addition, environmental factors, such as cigarette smoking, may cause
changes in the skin associated with aging. The language "treating
photodamage or aging" includes the alleviation, amelioration or
prevention of one or more symptoms of photodamage or aging of the skin.

[0065]Collagen is the main protein of connective tissue in animals and the
most abundant protein in mammals, making up about 25% of the total
protein content. It is a long, fibrous structural protein whose function
is quite different from those of globular proteins such as enzymes.
Strong, tough bundles of collagen called collagen fibers are a major
component of the extracellular matrix that supports most tissues and
gives cells structure from the outside, but collagen is also found inside
certain cells. Collagen has great tensile strength, and is the main
component of cartilage, ligaments, tendons, bone and teeth. Along with
soft keratin, it is responsible for skin strength and elasticity, and its
degradation leads to wrinkles that accompany aging

[0066]The language "modulate collagen levels" includes the adjusting
collagen levels or keeping collagen levels in proper measure or
proportion. In one embodiment, the collagen levels are increased,
elevated or stimulated. The language "an amount of a creatine compound
effective to modulate collagen levels" includes the amount of a creatine
compound necessary to increase, elevate of stimulate collagen levels.

[0067]In one embodiment, the amount of a creatine compound effective to
modulate collagen levels is at least between about 0.0001% to 10% by
weight. More particularly, the amount of a creatine compound effective to
modulate collagen levels may be about 0.0001%, 0.0002%, 0.0003%, 0.0004%,
0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%,
0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%, 0.009%, 0.01%, 0.02%,
0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and
10% by weight, or a range thereof. In a further embodiment, the amount of
a creatine compound effective to modulate collagen levels is at least
between about 0.001% and 1% by weight or between about 0.01% and 0.5% by
weight.

[0069]In another example, the creatine compounds, such as creatine
pyruvate, stimulate collagen levels by at least about 20% at a
concentration of about 1 μg/mL. Stimulation of collagen levels can be
determined by using the assay described in Example 2.

[0070]In one embodiment, the invention pertains to a method of treating
inflammation in a subject by administering an amount of a creatine
compound effective to modulate metalloproteinase. Inflammation is a
complicated biochemical response of the immune system to infection or
irritation and is characterized by redness, heat, swelling and pain.

[0071]Metalloproteinases (or metalloproteases) are a family of enzymes
from the group of proteinases. There are two subgroups of
metalloproteinases: metallocarboxypeptidases and metalloendopeptidases.
Proteinases can be divided into four families if characterized by the
nature of the most prominent functional group in their active site:
serine, cysteine, aspartic and metalloproteinases. Metalloproteinases
bind a metal ion such as Zn2+ or Ca2+ in their active site.
Important metalloproteinases are the bacterial enzyme thermolysin (which
is a metalloendopeptidase), the digestive enzymes carboxypeptidase A or B
(which are metallocarboxypeptidases), matrix metalloproteinases (MMP,
also metalloendopeptidases) and collagenases. Collagenases are a type of
metalloproteinases that break down the peptide bonds in collagen. MMPs
play an important role in tumor metastasis, embryonic development and
wound healing.

[0072]The language "modulate metalloproteinase" includes inhibiting or
stimulating the metalloproteinase activity. In one embodiment, the
metalloproteinase is inhibited.

[0073]In one embodiment, the amount of a creatine compound effective to
modulate metalloproteinase is at least between about 0.0001% to 10% by
weight. More particularly, the amount of a creatine compound effective to
modulate metalloproteinase may be about 0.0001%, 0.0002%, 0.0003%,
0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%,
0.003%, 0.004%, 0.005%, 0.005%, 0.006% 0.007%, 0.008%, 0.009%, 0.01%,
0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, and 10% by weight, or a range thereof. In a further embodiment, the
amount of a creatine compound effective to modulate metalloproteinase is
at least between about 0.001% and 1% by weight or between about 0.01% and
0.5% by weight.

[0075]In one embodiment, certain creatine compounds, such as creatine
ascorbate, inhibit metalloproteinase by at least about 10% at a
concentration of about 1 mg/mL. Methods for the determination of the
inhibition of metalloproteinase can be found in Example 6.

[0076]In another embodiment, the creatine compound inhibits
metalloproteinase by at least about 25% at a concentration of about 1
mg/mL of said creatine compound. In a further embodiment, the creatine
pyruvate.

[0078]In one embodiment, the creatine compound is administered topically
or orally. The term "topical administration" includes methods of delivery
such as laying on or spreading on the skin. It involves any form of
administration which involves the skin. Examples of compositions suitable
for topical administration, include but are not limited to, ointments,
lotions, creams, cosmetic formulations, and skin cleansing formulations.
Additional examples include aerosols, solids (such as bar soaps) and
gels.

[0079]The term "pharmaceutically acceptable" includes drugs, medicaments
or inert ingredients which are suitable for use in contact with the
tissues of humans and lower animals without undue toxicity,
incompatibility, instability, irritation, allergic response, and the
like, commensurate with a reasonable benefit/risk ratio. The term also
encompasses cosmetically acceptable ingredients.

[0080]The language "effective amount" is intended to include the amount of
the creatine compound sufficient to prevent, ameliorate or alleviate one
or more symptom that the administration of the creatine compound is
attempt to treat. An effective amount can be determined on an individual
basis and will be based, at least in part, on consideration of the
severity of the symptoms to be treated and the activity of the specific
analog selected if an analog is being used. Further, the effective
amounts of the creatine compound may vary according to the age of the
subject being treated. Thus, an effective amount of the creatine compound
can be determined by one of ordinary skill in the art employing such
factors as described above using no more than routine experimentation in
health care management.

[0081]The phrase "pharmaceutically acceptable carrier" includes a
pharmaceutically acceptable material, composition or vehicle, such as a
liquid or solid filler, diluent, excipient, solvent or encapsulating
material, involved in carrying or transporting a compound(s) of the
present invention within or to the subject such that it can performs its
intended function. Typically, such compounds are carried or transported
from one organ, or portion of the body, to another organ, or portion of
the body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can serve as
pharmaceutically acceptable carriers include: sugars, such as lactose,
glucose and sucrose; starches, such as corn starch and potato starch;
cellulose, and its derivatives, such as sodium carboxymethyl cellulose,
ethyl cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository waxes;
oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; glycols, such as propylene glycol;
polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;
esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,
such as magnesium hydroxide and aluminum hydroxide; alginic acid; fruit
acids, pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; phosphate buffer solutions; and other non-toxic compatible
substances employed in pharmaceutical formulations.

[0082]The topical pharmaceutical compositions of the present invention may
be made into a wide variety of product types. These include, but are not
limited to solutions, lotions, creams, beach products, gels, sticks,
sprays, pads, ointments, pastes, mousses and cosmetics. These product
types may comprise several types of carrier systems including, but not
limited to solutions, emulsions, gels and solids.

[0083]The topical pharmaceutical compositions of the present invention
formulated as solutions typically include a pharmaceutically-acceptable
aqueous or organic solvent. The terms "pharmaceutically-acceptable
aqueous solvent" and "pharmaceutically-acceptable organic solvent" refer
to a solvent which is capable of having dispersed or dissolved therein
the active compound, and possesses acceptable safety properties (e.g.
irritation and sensitization characteristics). Water is a typical aqueous
solvent. Examples of suitable organic solvents include: propylene glycol,
butylene glycol, polyethylene glycol (200-600), polypropylene glycol
(425-2025), glycerol, 1,2,4-butanetriol, sorbitol esters,
1,2,-6-hexanetriol, ethanol, isopropanol, butanediol, and mixtures
thereof. Preferably, these solutions contain from about 0.0001% to about
10% of the of the active compound (e.g., a creatine compound), more
preferably from about 0.001% to about 1%, and more preferably from about
0.01% to about 0.5%; and from about 1% to about 90% of an acceptable
aqueous or organic solvent, more preferably from about 1% to about 40%.

[0084]If the topical pharmaceutical compositions of the present invention
are formulated as an aerosol and applied to the skin as a spray-on, a
propellant is added to a solution composition. A more complete disclosure
of propellants useful herein can be found in Sagarin, Cosmetics Science
and Technology, 2nd Edition, Vol. 2, pp. 443-465 (1972).

[0085]Topical pharmaceutical compositions of the present invention may be
formulated as a solution comprising an emollient. An example of a
composition formulated in this way would be a sunscreen-containing
product. Preferably, such compositions contain from about 0.0001% to
about 10% of the active compound (e.g., a creatine compound), more
preferably from about 0.001% to about 1%, and more preferably from about
0.01% to about 0.5%; and from about 2% to about 90% of a topical
pharmaceutically-acceptable emollient.

[0086]The term "emollients" includes materials used for the prevention or
relief of dryness, as well as for the protection of the skin. A wide
variety of suitable emollients are known and may be used herein. Sagarin,
Cosmetics, Science and Technology, 2nd Edition, Vol. 1, p. 32-43 (1972),
incorporated herein by reference, contains numerous examples of suitable
materials.

[0087]A lotion can be made from a solution carrier system. Lotions
preferably comprise from 0.0001% to about 10% of the active compound
(e.g., a creatine compound), more preferably from about 0.001% to about
1%, and more preferably from about 0.01% to about 0.5%; from about 1% to
about 20%, preferably from about 5% to about 10%, of an emollient; and
from about 50% to about 90%, preferably from about 60% to about 80%,
water.

[0088]Another type of product that may be formulated from a solution
carrier system is a cream. A cream of the present invention would
preferably comprise from about 0.0001% to about 10% of the active
compound (e.g., a creatine compound), more preferably from about 0.001%
to about 1%, and more preferably from about 0.01% to about 0.5% of the
active compound; from about 5% to about 50%, preferably from about 10% to
about 20%, of an emollient, and from about 45% to about 85%, preferably
from about 50% to about 75%, water.

[0089]Yet another type of product that may be formulated from a solution
carrier system is an ointment. An ointment may comprise a simple base of
animal or vegetable oils or semi-solid hydrocarbons (oleaginous).
Ointments may also comprise absorption ointment bases which absorb water
to form emulsions. Ointment carriers may also be water soluble. An
ointment may also comprise from about 2% to about 10% of an emollient
plus from about 0.1% to about 2% of a thickening agent. A more complete
disclosure of thickening agents useful herein can be found in Segarin,
Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72-73 (1972),
incorporated herein by reference.

[0090]If the carrier is formulated as an emulsion, from about 1% to about
10%, preferably from about 2% to about 5%, of the carrier system
comprises an emulsifier. Emulsifiers may be nonionic, anionic or
cationic. Suitable emulsifiers are disclosed in, for example, U.S. Pat.
No. 3,755,560; U.S. Pat. No. 4,421,769; and McCutcheon's Detergents and
Emulsifiers, North American Edition, pages 317-324 (1986); the
disclosures of which are incorporated herein by reference. Preferred
emulsifiers are anionic or nonionic, although the other types may also be
used.

[0091]Lotions and creams can be formulated as emulsions as well as
solutions. Preferably such lotions comprise from about 0.0001% to about
10% of the active compound (e.g., a creatine compound), more preferably
from about 0.001% to about 1%, and more preferably from about 0.01% to
about 0.5% of the active compound; from about 1% to about 20%, preferably
from about 5% to about 10%, of an emollient; from about 25% to about 75%,
preferably from about 45% to about 95%, water; and from about 0.1% to
about 10%, preferably from about 0.5% to about 5%, of an emulsifier. Such
creams would preferably comprise from about 0.0001% to about 10% of the
active compound (e.g., a creatine compound), more preferably from about
0.001% to about 1%, and more preferably from about 0.01% to about 0.5% of
the active compound; from about 1% to about 20%, preferably from about 5%
to about 10%, of an emollient; from about 20% to about 80%, preferably
from about 30% to about 70%, water; and from about 1% to about 10%,
preferably from about 2% to about 5%, of an emulsifier.

[0092]Single emulsion skin care preparations, such as lotions and creams,
of the oil-in-water type and water-in-oil type are well-known in the
cosmetic art and are useful in the present invention. Multiphase emulsion
compositions, such as the water-in-oil-in-water type, as disclosed in
U.S. Pat. No. 4,254,105, incorporated herein by reference, are also
useful in the present invention. In general, such single or multiphase
emulsions contain water, emollients and emulsifiers as essential
ingredients.

[0093]Triple emulsion carrier systems comprising an
oil-in-water-in-silicone fluid emulsion composition as disclosed in U.S.
Pat. No. 4,960,764, incorporated herein by reference, are also useful in
the present invention. Preferably, this triple emulsion carrier system
can be combined with from about 0.0001% to about 10% of the active
compound (e.g., a creatine compound), more preferably from about 0.001%
to about 1%, and more preferably from about 0.01% to about 0.5% of the
active compound to yield the topical pharmaceutical composition of the
present invention.

[0094]Another emulsion carrier system useful in the topical pharmaceutical
compositions of the present invention is a micro-emulsion carrier system.
Such a system comprises from about 9% to about 15% squalane; from about
25% to about 40% silicone oil; from about 8% to about 20% of a fatty
alcohol; from about 15% to about 30% of polyoxyethylene sorbitan
mono-fatty acid (commercially available under the trade name Tweens) or
other nonionics; and from about 7% to about 20% water. This carrier
system is preferably combined with from about 0.0001% to about 10% of the
active compound (e.g., a creatine compound), more preferably from about
0.001% to about 1%, and more preferably from about 0.01% to about 0.5% of
the active compound.

[0095]If the topical pharmaceutical compositions of the present invention
are formulated as a gel or a cosmetic stick, a suitable amount of a
thickening agent, as disclosed supra, is added to a cream or lotion
formulation.

[0096]The topical pharmaceutical compositions of the present invention may
also be formulated as makeup products such as foundations.

[0097]The topical pharmaceutical compositions of the present invention may
also be. formulated as medicated pads. Suitable examples of these pads
are fully disclosed in U.S. Pat. Nos. 4,891,227 and 4,891,228, the
disclosures of which are incorporated herein by reference.

[0099]Various water-soluble materials may also be present in the
compositions of this invention. These include humectants, proteins and
polypeptides, preservatives and an alkaline agent. In addition, the
topical compositions herein can contain conventional cosmetic adjuvants,
such as dyes, opacifiers (e.g., titanium dioxide), pigments and perfumes.

[0100]The topical pharmaceutical compositions of the present invention may
also include a safe and effective amount of a penetration enhancing
agent. A preferred amount of penetration enhancing agent is from about 1%
to about 5% of the composition. Another useful penetration enhancer for
the present invention is the non-ionic polymer under the CTFA
designation: polyacrylamide and isoparrafin and laureth-7, available as
Sepigel from Seppic Corporation. Also useful is polyquatemium-32 and
mineral oil known as SalCare SC92 available from Allied Colloids,
Suffolk, Va. This is a class of cationic polymers which are generally
described in U.S. Pat. No. 4,628,078 and U.S. Pat. No. 4,599,379, both of
which are incorporated by reference herein.

[0101]Examples of useful penetration enhancers, among others, are
disclosed in U.S. Pat. No. 4,537,776; U.S. Pat. No. 4,552,872; U.S. Pat.
No. 4,557,934; U.S. Pat. No. 4,130,667; U.S. Pat. No. 3,989,816; U.S.
Pat. No. 4,017,641; and European Patent Application 0043738, the contents
of each of which are incorporated herein by reference.

[0102]Other conventional skin care product additives may also be included
in the compositions of the present invention. For example, collagen,
hyaluronic acid, elastin, hydrolysates, primrose oil, jojoba oil,
epidermal growth factor, soybean saponins, mucopolysaccharides, and
mixtures thereof may be used.

[0103]Various vitamins may also be included in the compositions of the
present invention. For example, Vitamin A, ascorbic acid, Vitamin B,
biotin, panthothenic acid, Vitamin D, Vitamin E and mixtures thereof and
derivatives thereof are contemplated.

[0104]Also contemplated are skin cleaning compositions comprising both
active compounds of the present invention and a cosmetically-acceptable
surfactant. The term "cosmetically-acceptable surfactant" includes a
surfactant which is not only an effective skin cleanser, but also can be
used without undue toxicity, irritation, allergic response, and the like.
Furthermore, the surfactant must be capable of being comingled with the
active compound in a manner such that there is no interaction which would
substantially reduce the efficacy of the composition.

[0105]The skin cleaning compositions of the present invention preferably
contain from 0.0001% to about 10% of the active compound (e.g., a
creatine compound), more preferably from about 0.001% to about 1%, and
more preferably from about 0.01% to about 0.5% of the active compound and
from about 1% to about 90%, more preferably from about 1% to about 10%,
of a cosmetically-acceptable surfactant.

[0106]The physical form of the skin cleansing compositions is not
critical. The compositions can be, for example, formulated as toilet
bars, liquids, pastes, mousses, or pads.

[0107]The surfactant component of the compositions of the present
invention are selected from anionic, nonionic, zwitterionic, amphoteric
and ampholytic surfactants, as well as mixtures of these surfactants.
Such surfactants are well-known to those skilled in the detergency art.

[0108]The cleaning compositions of the present invention can optionally
contain, at their art-established levels, materials which are
conventionally used in skin cleansing compositions.

[0109]Sunblocks and sunscreens incorporating creatine compounds are also
contemplated. The term "sun block" or "sun screen" includes compositions
which block UV light. Examples of sunblocks include, for example, zinc
oxide and titanium dioxide.

[0110]The combination of creatine compounds with a UVA and/or UVB
sunscreen would be advantageous. The inclusion of sunscreens in
compositions of the present invention will provide immediate protection
against acute UV damage.

[0112]Preferred sunscreens useful in the compositions of the present
invention are 2-ethylhexyl-p-methoxycinnamate,
butylmethoxydibenzoylmethane, 2-hydroxy-4-methoxybenzophenone,
octyldimethyl-p-aminobenzoic acid and mixtures thereof.

[0113]A safe and effective amount of sunscreen may be used in the
compositions of the present invention. The sunscreening agent must be
compatible with the active compound. Generally the composition may
comprise from about 1% to about 20%, preferably from about 2% to about
10%, of a sunscreening agent. Exact amounts will vary depending upon the
sunscreen chosen and the desired Sun Protection Factor (SPF).

[0114]Also particularly useful in the present invention are sunscreens
such as those disclosed in U.S. Pat. No. 4,937,370 and U.S. Pat. No.
4,999,186, incorporated herein by reference. The sunscreening agents
disclosed therein have, in a single molecule, two distinct chromophore
moieties which exhibit different ultra-violet radiation absorption
spectra. One of the chromophore moieties absorbs predominantly in the UVB
radiation range and the other absorbs strongly in the UVA radiation
range.

[0115]An agent may also be added to any of the compositions of the present
invention to improve the skin substantivity of those compositions,
particularly to enhance their resistance to being washed off by water, or
rubbed off. A preferred agent which will provide this benefit is a
copolymer of ethylene and acrylic acid. Compositions comprising this
copolymer are disclosed in U.S. Pat. No. 4,663,157 which is incorporated
herein by reference.

[0116]In another embodiment of the present invention, an anti-inflammatory
agent is included as an active agent along with the creatine compounds of
the invention. The anti-inflammatory agent protects strongly in the UVA
radiation range (though it also provides some UVB protection as well)
thereby preventing further skin damage caused by UV radiation, while the
creatine compounds of the invention treats existing damage. Thus the
combination provides broad protection. The topical use of
anti-inflammatory agents reduces photo-aging of the skin resulting from
chronic exposure to UV radiation. (See U.S. Pat. No. 4,847,071 and U.S.
Pat. No. 4,847,069 both of which are incorporated herein by reference.)

[0117]A safe and effective amount of an anti-inflammatory agent may be
added to the compositions of the present invention, preferably from about
0.1% to about 10%, more preferably from about 0.5% to about 5%, of the
composition. The exact amount of anti-inflammatory agent to be used in
the compositions will depend on the particular anti-inflammatory agent
utilized since such agents vary widely in potency.

[0119]A second class of anti-inflammatory agents which is useful in the
compositions of the present invention includes the nonsteroidal
anti-inflammatory agents. The variety of compounds encompassed by this
group are well-known to those skilled in the art. For detailed disclosure
of the chemical structure, synthesis, side effects, etc., of
non-steroidal anti-inflammatory agents, reference may be had to standard
texts, including Antiinflammatory and Anti-Rheumatic Drugs, K. D.
Rainsford, Vol. I-III, CRC Press, Boca Raton, (1985), and
Anti-inflammatory Agents. Chemistry and Pharmacology, 1, R. A. Scherrer,
et al., Academic Press, New York (1974). Mixtures of these non-steroidal
anti-inflammatory agents may also be employed, as well as the
pharmaceutically-acceptable salts and esters of these agents. For
example, etofenamate, a flufenamic acid derivative, is particularly
useful for topical application. Yet another class of anti-inflammatory
agents which are useful in the present invention are those disclosed in
U.S. Pat. No. 4,912,248, incorporated herein by reference. This patent
discloses compounds and diastereomeric mixtures of specific
2-naphthyl-containing ester compounds, especially naproxen ester and
naproxol ester compounds, having two or more chiral centers. Finally,
so-called "natural" anti-inflammatory agents are useful in the present
invention. For example, candelilla wax, alpha bisabolol, aloe vera,
Manjistha (extracted from plants in the genus Rubia, particularly Rubia
Cordifolia), and Guggal (extracted from plants in the genus Commiphora,
particularly Commiphora Mukul), may be used.

[0121]As set out above, certain embodiments of the present compounds can
contain a basic functional group, such as amino or alkylamino, and are,
thus, capable of forming pharmaceutically acceptable salts with
pharmaceutically acceptable acids. The term "pharmaceutically acceptable
salts" in this respect, includes the relatively non-toxic, inorganic and
organic acid addition salts of compounds of the present invention. These
salts can be prepared in situ during the final isolation and purification
of the compounds of the invention, or by separately reacting a purified
compound of the invention in its free base form with a suitable organic
or inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge
et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0122]In other cases, the compounds of the present invention may contain
one or more acidic functional groups and, thus, are capable of forming
pharmaceutically acceptable salts with pharmaceutically acceptable bases.
The term "pharmaceutically acceptable salts" in these instances includes
the relatively non-toxic, inorganic and organic base addition salts of
compounds of the present invention. These salts can likewise be prepared
in situ during the final isolation and purification of the compounds, or
by separately reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary amine.
Representative alkali or alkaline earth salts include the lithium,
sodium, potassium, calcium, magnesium, and aluminum salts and the like.
Representative organic amines useful for the formation of base addition
salts include ethylamine, diethylamine, ethylenediamine, ethanolamine,
diethanolamine, piperazine and the like.

[0123]The term "pharmaceutically acceptable esters" includes the
relatively non-toxic, esterified products of the compounds of the present
invention. These esters can be prepared in situ during the final
isolation and purification of the compounds, or by separately reacting
the purified compound in its free acid form or hydroxyl with a suitable
esterifying agent. Carboxylic acids can be converted into esters via
treatment with an alcohol in the presence of a catalyst. Hydroxyls can be
converted into esters via treatment with an esterifying agent such as
alkanoyl halides. The term is further intended to include lower
hydrocarbon groups capable of being solvated under physiological
conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See,
for example, Berge et al., supra.)

[0124]Wetting agents, emulsifiers and lubricants, such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release
agents, coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the compositions.

[0125]Formulations of the present invention include those suitable for
topical or oral, administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods well
known in the art of pharmacy. The amount of active ingredient which can
be combined with a carrier material to produce a single dosage form will
generally be that amount of the compound which produces a therapeutic
effect, as described herein.

[0126]Methods of preparing these formulations or compositions include the
step of bringing into association a compound of the present invention
with the carrier and, optionally, one or more accessory ingredients. In
general, the formulations are prepared by uniformly and intimately
bringing into association a compound of the present invention with liquid
carriers, or finely divided solid carriers, or both, and then, if
necessary, shaping the product.

[0128]Dosage forms for the topical or transdermal administration of a
compound of this invention include powders, sprays, ointments, pastes,
creams, lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a pharmaceutically
acceptable carrier, and with any preservatives, buffers, or propellants
which may be required.

[0130]Powders and sprays can contain, in addition to a compound of this
invention, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of these
substances. Sprays can additionally contain customary propellants, such
as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such
as butane and propane.

[0131]Transdermal patches have the added advantage of providing controlled
delivery of a compound of the present invention to the body. Such dosage
forms can be made by dissolving or dispersing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux of the
compound across the skin. The rate of such flux can be controlled by
either providing a rate controlling membrane or dispersing the active
compound in a polymer matrix or gel.

[0132]These compositions may also contain adjuvants such as preservatives,
wetting agents, emulsifying agents and dispersing agents. Prevention of
the action of microorganisms may be ensured by the inclusion of various
antibacterial and antifungal agents, for example, paraben, chlorobutanol,
phenol sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into the
compositions. In addition, prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents which
delay absorption such as aluminum monostearate and gelatin.

[0133]In some cases, in order to prolong the effect of a drug, it is
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a liquid
suspension of crystalline or amorphous material having poor water
solubility. The rate of absorption of the drug then depends upon its rate
of dissolution which, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug form is accomplished by dissolving or
suspending the drug in an oil vehicle.

[0134]The preparations of the present invention may be given topically or
orally. They are of course given by forms suitable for each
administration route. For example, they are administered in tablets or
capsule form, ointment; topical by lotion or ointment. Topical
administration is preferred.

[0135]Actual dosage levels of the active ingredients in the pharmaceutical
compositions of this invention may be varied so as to obtain an amount of
the active ingredient which is effective to achieve the desired
therapeutic response for a particular patient, composition, and mode of
administration, without being toxic to the patient.

[0136]The selected dosage level will depend upon a variety of factors
including the activity of the particular compound of the present
invention employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion of the
particular compound being employed, the duration of the treatment, other
drugs, compounds and/or materials used in combination with the particular
compound employed, the age, sex, weight, condition, general health and
prior medical history of the patient being treated, and like factors well
known in the medical arts.

[0137]The term "chronic treatment" includes continued treatment with a
creatine compound over an extended period during a subject's lifetime,
preferably for at least about three weeks, more preferably from about
three months to about twenty years, more preferably from about six months
to about ten years, more preferably still from about one year to about
five years.

[0138]While it is possible for a compound of the present invention to be
administered alone, it is preferable to administer the compound as a
pharmaceutical composition.

[0140]The creatine compound can be administered to the afflicted
individual alone or in combination with another creatine compound or
other agent. The other agents could be approved therapies, supplements
that protect against oxidative damage, energy enhancers, sugars,
intermediates of metabolism and nutrients among others. The creatine
compounds can be administered as pharmaceutically acceptable salts in a
pharmaceutically acceptable carrier. The compound may be administered to
the subject by a variety of routes, including, but not necessarily
limited to topical, oral (dietary), transdermal, or parenteral (e.g.,
subcutaneous, intramuscular, intravenous injection, bolus or continuous
infusion) routes of administration, for example. An effective amount
(i.e., one that is sufficient to produce the desired effect in an
individual) of a composition comprising a creatine analog is administered
to the individual. The actual amount of the creatine compound to be
administered will depend on factors such as the size and age of the
individual, in addition to the severity of symptoms, other medical
conditions and the desired aim of treatment. As discussed above,
preferably the compound is administered topically.

[0141]The creatine compound can be formulated according to the selected
route of administration (e.g., emulsion, solution, cream, powder, tablet,
capsule, transdermal patch). An appropriate composition comprising a
creatine analog can be prepared in a physiologically acceptable vehicle
or carrier. For example, a composition in tablet form can include one or
more additives such as a filler (e.g., lactose), a binder (e.g., gelatin,
carboxymethylcellulose, gum arabic), a flavoring agent, a coloring agent,
or coating material as desired. For solutions or emulsions in general,
carriers may include aqueous or alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral vehicles can
include sodium chloride, solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's or fixed oils. In addition, intravenous
vehicles can include fluid and nutrient replenishers, and electrolyte
replenishers, such as those based on Ringer's dextrose. Preservatives and
other additives can also be present. For example, antimicrobial,
antioxidant, chelating agents, and inert gases can be added. (See,
generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, Ed.,
1980).

[0142]The term "administration" is intended to include routes of
administration which allow the creatine compounds to perform their
intended function. Examples of routes of administration which may be used
include injection (topical, oral, subcutaneous, intravenous,
parenterally, intraperitoneally, inhalation, transdermal, and rectal.
Depending on the route of administration, the creatine compound may be
coated with or in a material to protect it from the natural conditions
which may detrimentally effect its ability to perform its intended
function. The administration of the creatine compound is done at dosages
and for periods of time effective to reduce, ameliorate or eliminate the
symptoms of aging. Dosage regimes may be adjusted for purposes of
improving the therapeutic or prophylactic response of the compound. For
example, several divided doses may be administered daily or the dose may
be proportionally reduced as indicated by the exigencies of the
therapeutic situation.

[0143]In one embodiment, the invention pertains, at least in part to a
packaged composition comprising an effective amount of a creatine
compound, a cosmetically acceptable carrier, and one or more cosmetic
adjuvants. In one embodiment, the effective amount of the creatine
compound is at least between about 0.0001 and 10% by weight, more
preferably between about 0.001 and 1% by weight, and even more preferably
between about 0.01 and 0.5% by weight.

[0144]In another embodiment, the packaged composition further comprises
instructions for use. In one embodiment, the instructions for use are
instructions for all the methods described herein.

[0145]Lipid peroxidation (the oxidative breakdown of polyunsaturated fatty
acids) is widely accepted as one of the general mechanisms of cellular
injury and death. The purpose of this example was to assess the ability
of creatine ascorbate, creatine monohydrate and creatine pyruvate to
function as free radical scavengers by measuring their inhibitory
activity against UV-induced lipid peroxidation, as compared to ascorbic
acid and magnesium ascorbyl phosphate.

[0147]FIG. 1 is a graph which illustrates the concentration dependent
effects of several creatine compounds, as well as ascorbic acid and
magnesium ascorbyl phosphate, on the inhibition of lipid peroxidation.
FIG. 1 indicates that creatine ascorbate (.diamond-solid.) exhibited the
best inhibition of lipid peroxidation (21% inhibition at 500 μg/mL)
when compared to the other creatine compounds tested. Creatine
monohydrate (.box-solid.) and creatine pyruvate (.tangle-solidup.)
exhibited less than 10% inhibitory activity at all concentrations tested.
Both ascorbic acid and magnesium ascorbyl phosphate had good inhibitory
activity (up to 49%), which demonstrated the technical success of the
assay.

[0148]Collagen secreted by dermal fibroblasts is a major component of the
extracellular matrix in the skin. In aged and photodamaged skin, the
level of new collagen is decreased due to the lower number and
deregulation of dermal fibroblasts. The purpose of this example was to
test creatine ascorbate (CA), creatine monohydrate (CM) and creatine
pyruvate (CP) on type I collagen levels in human dermal fibroblast
conditioned medium as compared with ascorbic acid (AA) and magnesium
ascorbyl phosphate (MAP).

Methods

[0149]Normal human dermal fibroblasts (passage 5, lot number 7F1245,
Cambrix, Walkersville, Md.) were seeded in a 96-well plate in DMEM medium
(high glucose) containing 5% fetal calf serum and grown to late
subconfluent stage. Two sets of aqueous solutions of 10 mg/mL of creatine
ascorbate, creatine monohydrate, creatine pyruvate, ascorbic acid and
magnesium ascorbyl phosphate were prepared in Type I water. The first set
of solutions were prepared immediately before being added to cell
cultures. The second set of solutions were preincubated at pH 4.0 (+0.1)
five days, then the solutions were lyophilized and the substrates were
redissolved in water at 10 mg/mL prior to being added to the cell
cultures. Both the freshly prepared solutions and the solutions of
preincubated test materials were administered to the cells, and cell
culture conditioned media was harvested 5 days after the start of the
experiment and tested for type I collagen by sandwich ELISA using
affinity purified antibodies, followed by streptavidin-avidin-HRP
conjugate and ABTS, according to standard ELISA protocol. The
colorimetric signal proportional to collagen content was measured with a
microplate spectrophotometer at 405 nm. For references describing this
method, see Dobek et al. "J. Dermatol. Sci." 8:18 (1994) and Zhao et al.
"Phytomedicine" 12:132 (2005).

Results

[0150]FIG. 2 illustrates the concentration-dependent effect of freshly
prepared test materials on the stimulation of type I collagen in human
dermal fibroblast conditioned medium. The control is shown as the dashed
line at 100%. As expected, there was a large increase in the levels of
type I collagen in the cell cultures exposed to all concentrations of
magnesium ascorbyl phosphate (MAP). Creatine ascorbate also significantly
increased the levels of type I collagen at concentrations of 10 μg/mL
and 100 μg/mL, although the amount of type I collagen expressed at the
highest concentration was significantly lower. Creatine monohydrate (CM)
and creatine pyruvate (CP) both moderately increased type I collagen
levels at concentrations of 10 μg/mL and 100 μg/mL, although at
1000 μg/mL, the creatine monohydrate and creatine pyruvate both were
better than creatine ascorbate and ascorbic acid:

[0151]FIG. 3 illustrates the concentration-dependent effect of
preincubated test materials on the stimulation of type I collagen in
human dermal fibroblast conditioned medium. The results are similar to
those shown in FIG. 3. The control is shown as the dashed line at 100%.

[0152]In conclusion, the results of both assays demonstrated the strong
stimulation of type I collagen by magnesium ascorbyl phosphate which
indicated the technical success of the experiment. Creatine ascorbate
showed a dose-dependent stimulation of type I collagen levels by about
50% above the non-treated control (water). This stimulation was
comparable to that of magnesium ascorbyl phosphate and better than
ascorbic acid in the 100 μg/mL concentration.

[0153]The purpose of this assay was to determine the effect creatine
ascorbate, creatine monohydrate and creatine pyruvate on the
mitochondrial metabolism on the entire cell culture using an MTT assay.
The MTT assay measures the activity of succinate dehydrogenase, a key
enzyme in the respiratory electron transport chain in mitochondia.

Methods

[0154]Two sets of aqueous solutions of 10 mg/mL of creatine ascorbate,
creatine monohydrate, creatine pyruvate, ascorbic acid and magnesium
ascorbyl phosphate were prepared in Type I water. The first set of
solutions were prepared immediately before being added to cell cultures.
The second set of solutions were preincubated at pH 4.0 (+0.1) five days,
then the solutions were lyophilized and the substrates were redissolved
in water at 10 mg/mL prior to being added to the cell cultures. Normal
human dermal fibroblasts (passage 5, lot number 7F1254, Cambrex,
Walkersville, Md.) were seeded in a 96-well plate in phenol red-free DMEM
medium (high glucose) containing 2.5% fetal calf serum at 2500 cells per
well and test materials, both freshly prepared solutions and preincubated
solutions, were added 24 hours later. At the end of the incubation period
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
added to the cell cultures and incubation was pursued for an additional
three hours. Culture media were then discarded, the cells were rinsed and
the intracellular MTT reduction product, formazan, was solubilized in
ethanol/acetic acid. The colorimetric signal proportional to the
mitochondrial activity in the cell cultures was measured with a
microplate spectrophotometer at 570 nm. For references describing this
method, see Berridge et al. "Arch. Biochem. Biophys." 303:474-482 (1993)
and Murimaki et al. "Biochem. Pharmacol." 44:2191-2197 (1992).

[0157]Because of their propensity to damage to vital biological systems,
reactive species, such as free radicals, have been implicated in aging
and in more than 100 disease. Production of oxygen reactive species is an
integral part of human metabolism is the skin. Furthermore, production of
oxygen reactive species is increased upon exposure to sunlight,
pollutants and during inflammation. The objective of this test was to
measure the antioxidant potential of creatine ascorbate, creatine
phosphate and creatine monohydrate using oxygen radical absorbance
capacity (ORAC) assay.

Methods

[0158]The ORAC assay was performed according to the method described by Ou
et al. "J. Agric. Food Chem." 49(10):4619-4626 (2001). The ORAC assay
measured the ability of antioxidant components to inhibit the decline in
disodium fluorescein (FL) fluorescence that is induced by the peroxyl
radical generator 2',2'-Azobis(2-amidinopropane)dihydrochloride (AAPH).
The reaction was conducted in a 96-well plate format. The reaction
mixture contained FL (6.3×10-7 M) and AAPH
(1.28×10-1 M) in phosphate-buffered saline (PBS). The final
dilution of test materials added (stock solutions at 1000 μg/mL, 100
μg/mL and 10 μg/mL) was 1/20. The reaction was started by the
addition of AAPH. Fluorescence was measured at the emission wavelength of
530 nm and excitation wavelength of 485 nm using a microplate
fluorimeter. ORAC values were calculated from the quantitation of the
areas under the FL decay curve and are expressed as micromole μmol
Trolox equivalents (TE) per mL.

[0160]The objective of this example was to determine whether creatine
monohydrate, creatine pyruvate and creatine ascorbate have an effect on
the expression of p53 in human dermal fibroblasts. P53 is a key protein
triggering genomic repair and apoptosis (programmed cell death) in
response to mutagenic stress such as UV irradiation and carcinogens.

Methods

A. Determination of Irradiation Dose

[0161]Human dermal fibroblasts were grown to subconfluence in phenol-free
DMEM medium (4 g/L glucose) supplemented with 5% serum and irradiated
with UVB light at 0.15 mW/cm2 for various periods of time. Cells
were than returned to incubator for 72 hours and cell numbers were
determined by sulforhodamine B method. For a description of this assay,
see Skehan et al. "J. Natl. Cancer Inst." 82:1107 (1990).

B. Effect of Test Materials on P53 Induction by UVB Irradiation

[0162]Confluent human dermal fibroblasts were trypsinized, counted and
plated at 750,000 cells/well in eight 6-well plates in phenol-free DMEM
medium (4 g/L glucose) supplemented with 5% serum. After three hours,
creatine ascorbate, creatine monohydrate, creatine pyruvate, ascorbic
acid and magnesium ascorbyl phosphate in a concentration of 50 μg/ml
were added and cells were incubated overnight at 37° C. in 5%
CO2 atmosphere. After 24 hours, p53 protein was induced by UVB
irradiation at 0.15 mW/cm2 for 120 and was allowed to accumulate for
4 hours. The cells were then lyzed and total p53 was quantified by
sandwich ELISA. The p53 values were standardized with regard to total
protein values determined with Bradford reagent. For a description of
this assay, see Carlisle et al. "Toxicological Sciences" 55:60 (2000).

Results

[0163]FIG. 7 illustrates the time (and thus dose) dependant effect of UVB
irradiation at 0.15 mW/cm2 on human dermal fibroblast proliferation.
This data indicated that after 55 minutes, the number of cells decreases
in a linear manner and that the number of cells decreased by about 35%. A
35% reduction is indicative of genomic damage strong enough to trigger
p53 upregulation, without compromising the cultures.

[0165]The objective of this example was to determine the effect of
creatine ascorbate, creatine monohydrate and creatine pyruvate on
metalloproteinase activity, compared with ascorbic acid and magnesium
ascorbyl phosphate.

Methods

[0166]Metalloproteinase (collagenase) activity was measured with Enzcheck
kit from Molecular Probes using quenched fluorescent gelatin and
Clostridium collagenase IV, a generic collagenase. Creatine ascorbate,
creatine monohydrate, creatine pyruvate, ascorbic acid and magnesium
ascorbyl phosphate, in concentrations of 1000 μg/mL, 100 μg/mL and
10 μg/mL, were incubated in the presence of a collagenase substrate
(quenched fluorescing-linked gelatin) and in the presence of the
proteolytic enzyme. Phenantroline, a potent metalloproteinase inhibitor
was used as a positive control. The kinetics of the release of the
digested, fluorescent gelatin were measured at excitation/emission
wavelengths of 485/530 nm.

[0168]Tyrosinase is believed to be a key enzyme in the melanin synthesis
pathway. The objective of this example was to assess the effect of
creatine ascorbate, creatine monohydrate and creatine pyruvate on
tyrosinase activity in vitro as compared with ascorbic acid and magnesium
ascorbyl phosphate.

Methods

[0169]Tyrosinase activity was measured according to the method described
by Pomerantz "Biochem. Biophys. Res. Commun." 16(2):188-194 (1964).
Mushroom tyrosinase stock solution was prepared in PBS at 2000 U/mL and
stored at 20° C. in 1 mL aliquots. Final concentration was 5
U/well (25 U/mL). Creatine ascorbate, creatine monohydrate, creatine
pyruvate, ascorbic acid and magnesium ascorbyl phosphate were dissolved
in PBS and further dilutions were made with Type I water. L-Dopa stock
solution was 20 mM. The tyrosinase substrate was prepared in PBS. The
assays were performed in a 96-well microtiter plates and read at 490 nm.

Results

[0170]The results indicated that creatine ascorbate exhibited a
significant inhibitory effect on the tyrosinase activity at
concentrations of 500 μg/mL and 50 μg/mL. At 5 μg/mL, inhibition
by creatine ascorbate was 26% compared to the control. Creatine ascorbate
inhibitory activity was lower than the activity of ascorbic acid and was
similar to the activity of magnesium ascorbyl phosphate. Creatine
pyruvate displayed a concentration dependent inhibitory effect, with 100%
inhibition at 500 μg/mL, 10% inhibition at 50 μg/mL and no
inhibition at 5 μg/mL.

[0171]The objective of this example was to compare the stability of
aqueous solutions of creatine ascorbate, creatine monohydrate and
creatine pyruvate, compared with ascorbic acid and magnesium ascorbyl
phosphate.

Methods

[0172]Aqueous solutions of 10 mg/mL of creatine ascorbate, creatine
monohydrate, creatine pyruvate, ascorbic acid and magnesium ascorbyl
phosphate were prepared in Type I water and brought to pH 4.0 (±0.1)
by adding acetic acid (creatine ascorbate, creatine monohydrate and
magnesium ascorbyl phosphate) or sodium hydroxide (ascorbic acid and
creatine pyruvate). The solutions were incubated for 5 days at room
temperature and were subsequently lyophilized, redissolved within 60
minutes of analysis and analyzed by isocratic HPLC on a C18 RF column
(250 mm×4.6 mm) at 1 mL/min. The chromatography conditions are
summarized in Table 1.

[0173]Creatine ascorbate and magnesium ascorbyl phosphate showed minor to
no degradation. Creatine monohydrate showed a clear degradation product,
and creatine pyruvate showed a 42% loss of the absorbance peak. Ascorbic
acid also showed degradation as indicated by a 28% decrease of
absorption.

EQUIVALENTS

[0174]Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, numerous equivalents to the
specific procedures described herein. Such equivalents are considered to
be within the scope of the present invention and are covered by the
following claims. The contents of all references, patents, and patent
applications cited throughout this application are hereby incorporated by
reference. The appropriate components, processes, and methods of those
patents, applications and other documents may be selected for the present
invention and embodiments thereof.